Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. As such, cell adhesion constitutes one of the fundamental mechanisms underlying numerous biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hematopoietic cells out of blood vessels and to the site of inflammatory injury. As such, cell adhesion plays a role in numerous pathologies such as, for example, inflammation and immune reactions in mammals.
Investigations into the molecular basis for cell adhesion have revealed that various cell-surface macromolecules—collectively known as cell adhesion molecules or receptors—mediate cell-cell and cell-matrix interactions. For example, proteins of the superfamily called “integrins” are key mediators in adhesive interactions between hematopoietic cells and their micro environment (M. E. Hemler, “VLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.”, Ann. Rev. Immunol., 8, p. 365 (1990)). Integrins are non-covalent heterodimeric complexes consisting of two subunits called α and β. There are at least 16 different “subunits (α1-α9, α-L, α-M, α-D, α-X, αIIB, α-V and α-E) and at least 9 different β (β1-β9) subunits which have been identified to date. Based on the type of its α and β subunit components, each integrin molecule can be categorized into a subfamily.
α4β1 integrin, also known as very late antigen of activation-4 (“VLA-4”) or CD49d/CD29, is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell-matrix adhesive interactions (M. E. Hemler, Ann. Rev. Immunol., 8, p. 365 (1990)). It serves as a receptor for the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-1 (“VCAM-1”), as well as to the extracellular matrix protein fibronectin (“FN”) (Ruegg et al., J. Cell Biol., 177, p. 179 (1991); Wayner et al., J. Cell Biol., 105, p. 1873 (1987); Kramer et al., J. Biol. Chem., 264, p. 4684 (1989); Gehlsen et al. Science, 24, p. 1228 (1988)). Anti-VLA4 monoclonal antibodies (“mAb's”) have been shown to inhibit VLA4-dependent adhesive interactions both in vitro and in vivo (Ferguson et al. Proc. Natl. Acad. Sci., 88, p. 8072 (1991); Ferguson et al., J. Immunol., 150, p. 1172 (1993)). Results of in vivo experiments suggest that the inhibition of VLA-4-dependent cell adhesion may prevent, inhibit or alter several inflammatory and autoimmune pathologies. (R. L. Lobb et al., “The Pathophysiologic Role of α4 Integrins In Vivo”, J. Clin. Invest., 94, pp. 1722-28 (1994)).
Another integrin, αIIbβIIIa integrin (“IIb/IIIa”), is the most abundant integrin found on the membrane surface of normal platelets. Jennings, et al., J. Biol. Chem., 257, p. 10458 (1982). Platelets depend on the adhesive interactions of glycoproteins, such as “IIb8IIIa integrin, for proper function. J. Hawiger, Atherosclerosis Reviews, 21, pp. 165-86 (1990). Thus, inhibition of this interaction is one method of regulating platelet thrombus formation or aggregation. A variety of compounds are known to inhibit αIIbβIIIa integrins from binding to their natural ligands and thereby can regulate human disorders associated with a hyperthrombotic state. Compounds known to inhibit IIb/IIIa are described in the following patents and patent applications: GB 2 271 567 A; GB 2 292 558 A; EP 0 645 376 A1; EP 0 668 278 A1; EP 0 608 759 A2; EP 0 635 492 A1; WO 94/22820; U.S. Pat. No. 5,340,798 and WO 94/09029; U.S. Pat. No. 5,256,812, EP 0 381 033 and U.S. Pat. No. 5,084,466; WO 94/18981; WO 94/01396 and U.S. Pat. No. 5,272,162; WO 94/21602; WO 94/22444; WO 94/29273; WO 95/18111; WO 95/18619; WO 95/25091; WO 94/18162, U.S. Pat. No. 5,220,050 and WO 93/16038; U.S. Pat. No. 4,879,313 and EP 0 352 249 B1; WO 93/16697, U.S. Pat. No. 5,227,490, EP 0 478 363 A2, U.S. Pat. No. 5,229,616 and WO 94/12181; U.S. Pat. No. 5,258,398 and WO 93/11759; WO 93/08181 and EP 0 537 980 A1; WO 93/09133; EP 0 530 505 B1; EP 0 566 919 A1; EP 0 540 334 B1; EP 0 560 730 A2; WO 93/10091, EP 0 542 363 A2 and WO 93/14077; EP 0 505 868 B1; EP 0 614 664 A1; U.S. Pat. No. 5,358,956; U.S. Pat. No. 5,334,596 and WO 94/26745; WO 94/12478; WO 94/14776; WO 93/00095; WO 93/18058, WO 93/07867, U.S. Pat. Nos. 5,239,113, 5,344,957 and EP 0 542 708 A1; WO 94/22825; U.S. Pat. No. 5,250,679 and WO 93/08174; U.S. Pat. No. 5,084,466; EP 0 668 278 A1; U.S. Pat. No. 5,264,420; WO 94/08962; EP 0 529 858; U.S. Pat. No. 5,389,631; WO 94/08577; EP 0 632 016; EP 0 503 548; EP 0 512 831 and WO 92/19595; WO 93/22303; EP 0 525 629; EP 0 604 800; EP 0 587 134; EP 0 623 615; EP 0 655 439; U.S. Pat. No. 5,446,056 and WO 95/14682; U.S. Pat. No. 5,399,585; WO 93/12074; EP 0 512 829; EP 0 372 486 and U.S. Pat. No. 5,039,805; EP 0 632 020 and U.S. Pat. No. 5,494,922; U.S. Pat. No. 5,403,836; WO 94/22834; WO 94/21599; EP 0 478 328; WO 94/17034 WO 96/20192, WO 96/19223, WO 96/19221, WO 96/19222, EP 727425, EP 478362, EP 478363, U.S. Pat. Nos. 5,272,158, 5,227,490, 5,294,616, 5,334,596, EP 645376, EP 711770, U.S. Pat. No. 5,314,902, WO 94/00424, U.S. Pat. No. 5,523,302, EP 718287, DE 4446301, WO 96/22288, WO 96/29309, EP 719775, EP 635492, WO 96/16947, U.S. Pat. No. 5,602,155, WO 96/38426, EP 712844, U.S. Pat. No. 5,292,756, WO 96/37482, WO 96/38416, WO 96/41803, WO 97/11940
Each of these references is specifically incorporated herein in its entirety.
In order to identify the minimum active amino acid sequence necessary to bind VLA-4, Komoriya et al. synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin. (“The Minimal Essential Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within the Alternatively Spliced Type III Connecting Segment Domain of Fibronectin Is Leucine-Aspartic Acid-Valine”, J. Biol. Chem., 266 (23), pp. 15075-79 (1991)). They identified an 8-amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val and Leu-Asp-Val-Pro-Ser, that possessed inhibitory activity against FN-dependent cell adhesion. These results suggested that the tripeptide Leu-Asp-Val was the minimum sequence for cell-adhesion activity. It was later shown that Leu-Asp-Val binds only to lymphocytes that express an activated form of VLA-4, thus casting doubt on the utility of such a peptide in vivo (E. A. Wayner et al., “Activation-Dependent Recognition by Hematopoietic Cells of the LDV Sequence in the V Region of Fibronectin”, J. Cell. Biol., 116(2), pp. 489-497 (1992)). However, certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo (T. A. Ferguson et al., “Two Integrin Binding Peptides Abrogate T-cell-Mediated Immune Responses In Vivo”, Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991); and S. M. Wahl et al., “Synthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitment”, J. Clin. Invest., 94, pp. 655-62 (1994)).
A cyclic pentapeptide, Arg-Cys-Asp-TPro-Cys(wherein TPro denotes 4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to FN has also been described. (See, e.g., D. M. Nowlin et al. “A Novel Cyclic Pentapeptide Inhibits α4β1 and α5β1 Integrin-mediated Cell Adhesion”, J. Biol. Chem., 268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862). This pentapeptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a common motif in the recognition site for several extracellular-matrix proteins.
Examples of other VLA-4 inhibitors have been reported, for example, in copending U.S. patent application Ser. No. 08/376,372, specifically incorporated by reference herein. U.S. Ser. No. 376,372 describes linear peptidyl compounds containing b-amino acids which have cell adhesion inhibitory activity. International patent applications WO 94/15958 and WO 92/00995, specifically incorporated by reference, describe cyclic peptide and peptidomimetic compounds with cell adhesion inhibitory activity. International patent applications WO 93/08823 and WO 92/08464 (specifically incorporated by reference herein) describe guanidinyl-, urea- and thiourea-containing cell adhesion inhibitory compounds. U.S. Pat. No. 5,260,277 describes guanidinyl cell adhesion modulation compounds, and is also specifically incorporated herein.
Despite these advances, there remains a need for small, potent inhibitors of cell adhesion, particularly for potent inhibitors of VLA-4 or IIb/IIIa cell adhesion. Ideally, such inhibitors would be small so that they may be administered orally. Such compounds would provide useful agents for treatment, alteration, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 or IIb/IIIa binding.